The long-term objective of this proposal is to enhance our understanding of cellular mechanisms involved in canalicular bile formation and cholestasis. Specific aims are as follows: 1) Evaluate mechanisms involved in the regulation of hepatocyte Na+/H+ exchange by studying the effects of intracellular Ca++, cyclic AMP and portein kinase C on H+=efflux, intracellular pH and Na+ uptake in isolated rat hepatocytes. 2) Determine the role of Na+/H+ exchange in canalicular bile formation by studying the effect of this exchange on biliary HC03- excretion in perfused rat livers. 3) Further evaluate the role of intracellular Ca++ in bile acid-induced cholestasis. The effects of cholestatic bile acids on Ca++-dependent degradative enzymes, like phospholipases and proteases, will be examined in isolated hepatocytes. 4) Determine the role of inorganic anion exchange in hepatic organic anion excretion by studying pH-gradient depenent transport of DIDS (diisothiocyanostilbene disulfonic acid), S04= and Cl- isolated plasma membrane vesicles. Established techniques will be used to study solute transport (Na+, C1-, S04= and DIDS), manipulate intracellular pH (NH4C1 and C02 pulse techniques), and determine intracellular phospholipid and protein hydrolysis. Fluorescence techniques will be used to determine intracellular (Ca++) (quin-2 and fura-2) and pH (BCECF). Various agents will be used to increase intracellular (Ca++) (phenylephrine, arginine-vasopressin & Ca++-ionophores), buffer intracellular Ca++ (MAPTAM), mimic cAMP (dibutyryl cAMP & 8-bromo-cAMP), activate protein kinase C (phorbol esters & diacylglycerols), inhibit the Ca++-calmodulin system (trifluoperazine and calmidazolium), and stimulate biliary HCO3-excretion (ursodeoxycholate and norchenodeoxycholate). Apart from increasing our understanding of bile formation and cholestasis, successful completion of these studies should also provide valuable information regarding the roles of second messengers in the regulation of intracellular pH, hepatocellular growth and regeneration, as well as information regarding a common mechanism for hepatic organic anion excretion.